Prostanoid formation in primary astroglial cell cultures: Ca-dependency and stimulation by A 23187, melittin and phospholipases A2 and C

Prostaglandin (PG) and thromboxane B₂ (TXB₂) biosynthesis was studied in cultured astrocytes from neonatal rat brain hemispheres. After two weeks of cultivation, prostanoids were formed with the spectrum: PGD₂ > TXB₂ > PGF₂ > PGE₂, as measured by specific radioimmunoassays. Under basal conditions PGD₂ biosynthesis (9.55 ng/mg protein/15 min) was in the same order of magnitude as the sum of the other prostanoids. The formation of prostanoids was stimulated in a concentration dependent manner (up to 6–10 fold) by the calcium ionophore A 23187 (0.01–10 μM) as well as by melittin (0.01–5 μg/ml), phospholipase A₂ (10–40 U/ml) and phospholipase C (0.01–1 U/ml). Basal and evoked PG and TXB₂ biosynthesis depended on the availability of Ca²⁺, as demonstrated in Ca²⁺ free incubation medium containing Na₂EDTA (1 μM), or with verapamil (100 μM) and 3,4,5-trimethoxybenzoic acid-8-(diethylamino)-octylester-HCl (TMB-8, 1–100 μM). Indomethacin (10 μM), mepacrine (100 μM) and p-bromophenacylbromide (50 μ M) inhibited basal and evoked PG formation. Thin-layer chromatography (TLC) detection after incubation of the cells with [³H]arachidonic acid (1 μCi/ml, for 60 min) confirmed the results obtained by radioimmunoassay. Incubation of [³H]arachidonic acid labelled cells with inonophore or phospholipases, followed by lipid extraction and TLC, showed that A 23187 liberated [³H]arachidonic acid predominantly from phosphatidylethanolamine, whereas phospholipase A₂ and C reduced mainly the labelling of the phosphatidyl-inositol/-choline fraction. Potassium depolarization of the cells did not enhance prostanoid formation. Similarly, drugs with affinity to - or β-adrenoceptors, or to dopamine-, 5-hydroxytryptamine-, muscarine-, histamine-, glutamate-, aspartate-, GABA, adenosine- and opioid-receptors failed to stimulate prostanoid biosynthesis. Also compounds like angiotensin, bradykinin and thrombin were ineffective in this respect.

In conclusion, our results confirm that cultured astrocytes possess the complete pattern of enzymes necessary for prostanoid formation and hence might play a crucial role in brain prostanoid biosynthesis. Stimulation of prostanoid biosynthesis involves Ca²⁺-dependent activation of phospholipase A₂, cyclooxygenase reaction and further PG metabolism. However, the endogenous stimulus for enhanced prostanoid synthesis in the brain still has to be established.     

Meiotic cohesin STAG3 is required for chromosome axis formation and sister chromatid cohesion

The cohesin complex is essential for mitosis and meiosis. The specific meiotic roles of individual cohesin proteins are incompletely understood. We report in vivo functions of the only meiosis‐specific STAG component of cohesin, STAG3. Newly generated STAG3‐deficient mice of both sexes are sterile with meiotic arrest. In these mice, meiotic chromosome architecture is severely disrupted as no bona fide axial elements (AE) form and homologous chromosomes do not synapse. Axial element protein SYCP3 forms dot‐like structures, many partially overlapping with centromeres. Asynapsis marker HORMAD1 is diffusely distributed throughout the chromatin, and SYCP1, which normally marks synapsed axes, is largely absent. Centromeric and telomeric sister chromatid cohesion are impaired. Centromere and telomere clustering occurs in the absence of STAG3, and telomere structure is not severely affected. Other cohesin proteins are present, localize throughout the STAG3‐devoid chromatin, and form complexes with cohesin SMC1β. No other deficiency in a single meiosis‐specific cohesin causes a phenotype as drastic as STAG3 deficiency. STAG3 emerges as the key STAG cohesin involved in major functions of meiotic cohesin.     

The Montreal Cognitive Assessment (MoCA) - A Sensitive Screening Instrument for Detecting Cognitive Impairment in Chronic Hemodialysis Patients

Background

Chronic kidney disease (CKD) patients undergoing hemodialysis (HD) therapy have an increased risk of developing cognitive impairment and dementia, which are known relevant factors in disease prognosis and therapeutic success, but still lack adequate screening in clinical routine. We evaluated the Montreal Cognitive Assessment (MoCA) for suitability in assessing cognitive performance in HD patients in comparison to the commonly used Mini-Mental State Examination (MMSE) and a detailed neuropsychological test battery, used as gold standard.

Methods

43 HD patients and 42 healthy controls with an average age of 58 years, were assessed with the MoCA, the MMSE and a detailed neuropsychological test battery, covering the domains of memory, attention, language, visuospatial and executive functions. Composite scores were created for comparison of cognitive domains and test results were analyzed using Spearman''s correlation and linear regression. Cognitive dysfunction was defined using z-score values and predictive values were calculated. Sensitivity and specificity of the MoCA were determined using receiver operating characteristic (ROC) analysis.

Results

HD patients performed worse in all cognitive domains, especially in memory recall and executive functions. The MoCA correlated well with the detailed test battery and identified patients with cognitive impairment with a sensitivity of 76.7% and specificity of 78.6% for a cut-off value of ≤24 out of 30 points. In the detailed assessment executive functions accounted significantly for performance in the MoCA. The MMSE only discriminated weakly between groups.

Conclusions

The MoCA represents a suitable cognitive screening tool for hemodialysis patients, demonstrating good sensitivity and specificity levels, and covering executive functions, which appear to play an important role in cognitive performance of HD patients.     

Improvement of homologous GH10 xylanase production by deletion of genes with predicted function in the Aspergillus nidulans secretion pathway

Filamentous fungi are important cell factories for large-scale enzyme production. However, production levels are often low, and this limitation has stimulated research focusing on the manipulation of genes with predicted function in the protein secretory pathway. This pathway is the major route for the delivery of proteins to the cell exterior, and a positive relationship between the production of recombinant enzymes and the unfolded protein response (UPR) pathway has been observed. In this study, Aspergillus nidulans was exposed to UPR-inducing chemicals and differentially expressed genes were identified by RNA-seq. Twelve target genes were deleted in A. nidulans recombinant strains producing homologous and heterologous GH10 xylanases. The knockout of pbnA (glycosyltransferase), ydjA (Hsp40 co-chaperone), trxA (thioredoxin) and cypA (cyclophilin) improved the production of the homologous xylanase by 78, 171, 105 and 125% respectively. Interestingly, these deletions decreased the overall protein secretion, suggesting that the production of the homologous xylanase was specifically altered. However, the production of the heterologous xylanase and the secretion of total proteins were not altered by deleting the same genes. Considering the results, this approach demonstrated the possibility of rationally increase the production of a homologous enzyme, indicating that trxA, cypA, ydjA and pbnA are involved in protein production by A. nidulans.     

Siderophores drive invasion dynamics in bacterial communities through their dual role as public good versus public bad

Microbial invasions can compromise ecosystem services and spur dysbiosis and disease in hosts. Nevertheless, the mechanisms determining invasion outcomes often remain unclear. Here, we examine the role of iron-scavenging siderophores in driving invasions of Pseudomonas aeruginosa into resident communities of environmental pseudomonads. Siderophores can be ‘public goods’ by delivering iron to individuals possessing matching receptors; but they can also be ‘public bads’ by withholding iron from competitors lacking these receptors. Accordingly, siderophores should either promote or impede invasion, depending on their effects on invader and resident growth. Using supernatant feeding and invasion assays, we show that invasion success indeed increased when the invader could use its siderophores to inhibit (public bad) rather than stimulate (public good) resident growth. Conversely, invasion success decreased the more the invader was inhibited by the residents’ siderophores. Our findings identify siderophores as a major driver of invasion dynamics in bacterial communities under iron-limited conditions.     

Hyperostotic fish bones (“Tilly bones”) from presumably Pliocene phosphorites of the Lake Manyara area,northern Tanzania

PalZ - Hyperostotic fish bones from the presumable Pliocene phosphate-bearing deposits along Lake Manyara, Tanzania, are described. In contrast to marine occurrences of this kind, the pathological...     

Litter decomposing and ectomycorrhizalBasidiomycetes in an igapó forest

An igapó forest near the confluence of Rio Tarumã Mirim (Tarumãzinho) and Rio Negro has been studied. It is a typical ectotroph forest with a raw humus layer and suppressed litter decomposing activity by Higher (i.e., carpophore-producing) Fungi. The number of the latter is about one-fifth of that observed in the (anectotrophic) terra firme forest. All ectotrophically mycorrhizal fungi observed belonged in three families:Amanitaceae, Boletaceae, Russulaceae. Leguminosae are dominant, and of theseAldina latifolia andSwartzia cf.polyphylla were demonstrably ectomycorrhizal. The scarcity of mineral nutrients in the soils of igapó, campinarana and campina is overcome by direct cycling through ectomycorrhizae. This is in contrast to other black- and white-water inundated forest communities in Amazonia.Litter Decomposition and Ectomycorrhiza in Amazonian Forests3. Previous contributions seeSinger & Araujo (1979) andSinger & al. (1983).     

Influence of hormones and drugs on glutathione-S-transferase levels in primary culture of adult rat hepatocytes

GST activities against 1-Chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) were measured in isolated and cultured adult rat hepatocytes. Within 24 h in culture, both GST activities decreased to about 70% and either stabilized at this level (CDNB) or recovered (DCNB) to the initial level. Use of hyaluronidase in addition to collagenase during the isolation of the cells strongly reduced both activities and its stimulation by various drugs for up to 168 h. The hormones insulin, glucagon, triiodothyronine, estradiol, testosterone, and progesterone did not affect GST activity, while dexamethasone showed some interference. In the presence of dexamethasone the activity against CDNB was mainly stimulated by the combination of methylcholanthrene (MC) and phenobarbital (PB) to about 260% within 168 h. The activity against DCNB was stimulated predominantly by MC alone reaching 170% after 168 h. Quantification of the GST subunits Ya, Yb₁ and Yp by an ELISA technique revealed a strong decrease of Ya, a transient increase of Yb₁ after 24 h followed by a moderate decrease, and a stable low level of the transformation marker Yp during cultivation. The level of Ya was markedly induced by PB, particularly in combination with MC. The level of Yb₁ was equally induced by MC or PB with no synergistic effect. Yp was not affected by these drugs. None of the hormones affected the level of these GST subunits. These results indicate that the physiological type of regulation of the GSTs is maintained during primary culture and no signs of dedifferentiation or transformation are observed. Furthermore, they demonstrate that the interaction of drugs and hormones and their inducing potential can be efficiently studied in the cultured hepatocytes.Abbreviations ABTS 2,2-Azino-bis(3-ethylbenzthiazoline-6-sulfonate) - CDNB I-Chloro-2,4-dinitrobenzene - DCNB 1,2-dichloro-4-nitrobenzene; DEX, dexamethasone - DMSO dimethylsulfoxide - GST glutathione Stransferase - MC methylcholanthrene - N, NIC nicotinamide - -NF -naphthoflavone - PB phenobarbital - PBS phosphate buffered saline     

Helospectin-like immunoreactivity in the esophagus

Helospectin I and II are two non-amidated, VIP-like peptides, isolated from the salivary gland venom of the lizard Heloderma horridum. The lower esophagus of cat, sheep and man was analyzed for helospectin-like immunoreactivity.

Immunocytochemistry revealed helospectin-immunoreactive nerve fibers in the muscle layers, submucosa and mucosa of all species studied. In myenteric ganglia helospectin-immunoreactive nerve fibers and nerve cell bodies could be seen. Double immunostaining for helospectin and vasoactive intestinal peptide (VIP) revealed their coexistence in nerve fibers and cell bodies throughout the lower esophagus of all species tested. Double immunostaining for helospectin and neuropeptide Y revealed their coexistence in nerve fibres surrounding vascular and non-vascular smooth muscle. In the cat and sheep (but not in man) a subpopulation of the helospectin/VIP-containing fibers stored, in addition, substance P.

The helospectin-immunoreactive material in the esophagus probably constitutes a novel neuropeptide. The distribution of the VIP/helospectin-immunoreactive neurons and fibers indicates their possible involvement in the regulation of motor and secretory activities.     

Stigmatella aurantiaca Sg a15 carries genes encoding type I and type II 3-deoxy-d-arabino-heptulosonate-7-phosphate synthases: involvement of a type II synthase in aurachin biosynthesis

3-Deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthases catalyse the first step of the shikimate pathway. Two unrelated DAHP synthase types have been described in plants and bacteria. Two type II (aroA(A2) and aroA(A5)) and one type I DAHP synthase gene (aroA001) were identified from the myxobacterium Stigmatella aurantiaca Sg a15. Inactivation of aroA(A5) leads to a mutant that is impaired in the biosynthesis of aurachins, which are electron transport inhibitors and contain an anthranilate moiety. Feeding of anthranilic acid to the mutant culture restores production of aurachins. Inactivation of aroA(A2) and aroA001 does not impair production of aurachins or other known secondary metabolites of S. aurantiaca Sg a15.